Hypoxia-induced 15-hydroxyeicosatetraenoic acid (15-HETE) is an essential mediator to constrict pulmonary arteries (PA). The signaling pathway involved in 15-HETE-induced PA vasoconstriction remains obscure. The aim of the present study was to test the hypothesis that hypoxic PA constriction induced by 15-HETE was possibly regulated by the extracellular signal-regulated kinase-1/2 (ERK1/2) pathway. PA ring tension measurement, Western blot and immunocytochemistry were used in the study to determine the possible role of ERK1/2 in 15-HETE-induced PA vasoconstriction. The organ bath for PA rings tension study was employed. Adult male Wistar rats were raised in hypoxic environment with fractional inspired oxygen (FIO2, 0.12) for 9 d. PA 1~1.5 mm in diameter were dissected and cut into 3 mm long rings for tension study. ERK1/2 up-stream kinase (MEK) inhibitor PD98059, which blocks the activation of ERK1/2, was used. The results showed that pretreatment of PD98059 significantly blunted 15-HETE-induced PA vasoconstrictions in the rings from hypoxic rat. Moreover, in endothelium-denuded rings, PD98059 also significantly attenuated 15-HETE-induced vasoconstriction. Phosphorylation of ERK1/2 in pulmonary arterial smooth muscle cells (PASMCs) of rat was enhanced evidently when stimulated by 15-HETE. Thus, the data suggest that ERK1/2 signaling pathway is involved in 15-HETE-induced hypoxic pulmonary vasoconstriction.
Animals ; Flavonoids ; pharmacology ; Hydroxyeicosatetraenoic Acids ; antagonists & inhibitors ; pharmacology ; Hypoxia ; physiopathology ; MAP Kinase Signaling System ; physiology ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; drug effects ; Pulmonary Artery ; cytology ; drug effects ; physiopathology ; Rats ; Rats, Wistar ; Vasoconstriction ; drug effects

15-hydroxyeicosatetraenoic acid (15-HETE) plays an important role in hypoxia-induced pulmonary vasoconstriction. Release of nitric oxide (NO) is apparently decreased and activity of endothelial nitric oxide synthase (eNOS) is impaired in chronic hypoxia. However, little is known whether 15-HETE contributes to eNOS/NO pathway in the constriction induced by 15-HETE. We examined the response of rat pulmonary artery (PA) rings to 15-HETE, the production of NO, total eNOS expression and the phosphorylation of eNOS in bovine pulmonary artery endothelial cells (BPAECs) stimulated by 15-HETE. Rat PA rings were divided into three groups: endothelium intact group, endothelium denuded group, and nitro-L-arginine methyl ester (L-NAME, 0.1 mmol/L, an inhibitor of eNOS) group. Constrictions to 15-HETE were significantly enhanced in endothelium denuded group and L-NAME group (both P< 0.05 vs endothelium intact group, n= 9); BPAECs were incubated in different conditions to test nitrite production by Greiss method. Nitrite production was significantly reduced by 1 mumol/L 15-HETE (P<0.05), and increased by the lipoxygenase inhibitors, 10 mumol/L cinnamyl 3,4- dihydroxy-[alpha] -cyanocinnamate (CDC, P< 0.05) and 0.1 mmol/L nordihydroguiairetic acid (NDGA, P< 0.01 ); Western blot analysis of extracts from BPAECs incubated with 15-HETE in different time was carried out to test total eNOS expression, and the expression was changed unobviously. Immunoprecipitation (IP) and Western blot analysis of cell extracts from BPAECs treated with 2 mumol/L 15-HETE in different length of time were accomplished, using phospo-eNOS-threonine 495 (Thr495, an inhibitory site) antibody for IP, and eNOS or 15-lipoxygenase (15-LO) antibodies for Western blot. 15-HETE depressed eNOS activity by increasing the levels of phospho-eNOS-Thr 495. The data suggest that eNOS/NO pathway is involved in PA constrictions induced by 15-HETE and that 15-HETE depresses eNOS activity by phosphorylation in Thr495 site. The protein interaction between phospho-eNOS (Thr495) and 15-LO is discovered for the first time.
Animals ; Cattle ; Down-Regulation ; drug effects ; Endothelium, Vascular ; cytology ; drug effects ; enzymology ; Hydroxyeicosatetraenoic Acids ; pharmacology ; In Vitro Techniques ; Male ; Nitric Oxide Synthase Type III ; metabolism ; Pulmonary Artery ; cytology ; enzymology ; physiology ; Rats ; Rats, Wistar

AIMTo observe the effect of subtypes of Kv channels in rat pulmonary artery smooth muscle cells (PASMCs) on the process of pulmonary vasoconstriction induced by 15-HETE.

METHODSIn the present study, ring of rabbit PA with specific Kv channel blockers were employed to functionally identify certain channel subtypes that took part in the process of 15-HETE induced pulmonary vasoconstriction; RT-PCR and Western blotting analysis were also used to measure the expression of subtypes of Kv in PASMCs exposed to 15-HETE,chronic hypoxia.

RESULTSBlocking of Kv1. 1, Kv1. 2, Kv1. 3 and Kv1. 6 channels did not affect 15-HETE induced vasoconstriction in normoxic rats; 15-HETE did not affect expression of Kv1. 1 and Kv1. 2 channels; 15-HETE significantly downregulated the expression of mRNA and protein of Kv1. 5 and Kv2. 1 in rat PASMCs.

CONCLUSIONThe results suggested that hypoxia may block Kv1. 5 and Kv2. 1 channels via 15-HETE mediated mechanism, leading to decrease numbers of functional Kv1. 5 and Kv2. 1 channels in PASMCs, leading to PA vasoconstriction.

Animals ; Cell Hypoxia ; Cells, Cultured ; Hydroxyeicosatetraenoic Acids ; pharmacology ; Hypoxia ; physiopathology ; Kv1.5 Potassium Channel ; biosynthesis ; genetics ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; metabolism ; Potassium Channels, Voltage-Gated ; antagonists & inhibitors ; Pulmonary Artery ; physiopathology ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Shab Potassium Channels ; biosynthesis ; genetics ; Vasoconstriction ; drug effects

OBJECTIVETo study osteoblastic phenotype expression of New Zealand rabbit skin fibroblasts transfected with mouse core binding factor a1/osteoblast specific transplanting factor-2 gene (Cbfa1/Osf2).

METHODSCbfa1/Osf2 gene, engineered into eukaryotic expression vector pSG5, was introduced into New Zealand rabbit skin fibroblasts with catholyte liposomes-Lipofectamine 2000. Meanwhile, those transfected pSG5 and un-transfected were set the control groups. The expression of Cbfa1 gene, osteocalcin (OCN) gene, alkaline phosphatase (ALP) gene and pre-peptide 2 alpha gene of collagen type I were detected by RT-PCR assay. Cbfa1 protein was detected by Western-Blot assay, in-cell ALP activity by p-nitrophenyl phosphate (PNPP) assay and OCN content in the supernatant by radio-immunity method. The ossification nodules was detected by Alizarin-Red staining and scanning electron microscope.

RESULTSCbfa 1mRNA and Cbfa1 protein were expressed in New Zealand rabbit skin fibroblasts transfected with pSG5-Cbfa1/Osf2 from the first day to the fifth day, but they were not detected in the control groups. In the pSG5-Cbfa1/Osf2 transfected group, the expression of ALP gene and OCN gene were respectively induced from the third day and the forth day, pre-peptide 2 alpha gene of collagen type I was enhanced from the third day. From the sixth day, ALP activity greatly increased, OCN strongly secreted, and they were maintained at a high level for about 4 weeks, and the difference was significant compared with the control group (P < 0.05). On the forty-second day, ossification nodules were found on the surface of pSG5-Cbfa1/Osf2 gene transfected cells.

CONCLUSIONSNew Zealand rabbit skin fibroblasts transfected with pSG5-Cbfa1/Osf2 can express osteogenesis-related genes and proteins, and form ossification nodules on their surface.

Alkaline Phosphatase ; biosynthesis ; genetics ; Animals ; Cell Adhesion Molecules ; biosynthesis ; genetics ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; biosynthesis ; genetics ; Fibroblasts ; cytology ; physiology ; Gene Expression ; Genetic Vectors ; Mice ; Osteocalcin ; biosynthesis ; genetics ; Osteogenesis ; genetics ; physiology ; Rabbits ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo investigate bone defect healing by true bone ceramic complex carrying core binding factor a1 (Cbfa1) gene modified rabbit skin fibroblasts.

METHODSTransfect rabbit skin fibroblasts (RSF) with both eukaryotic expression vector pSG5 which could express Cbfa1 gene and pSG5. After being cultured for 48 h, the transfected RSF were seeded into true bone ceramic (TBC) of 2 cm in length and 4 mm in diameter to construct pSG5-Cbfa1/RSF/TBC complex and pSG5/RSF/TBC complex. Forty-eight bone defect model rabbits were randomized into four groups, each has 6 rabbits (12 radius), due to different treatment. group I: with pSG5-Cbfa1/RSF/TBC complex, group II: with pSG5/RSF/TBC complex, group III: with TBC, Group IV: empty control. After being seeded and cultured for about 24 h the complexes were implanted into 2 cm long bone defects in the middle of bilateral radius of rabbits. The radius were inspected by X-ray and then the specimens were collected at the end of the fourth and twelfth weeks after operation. Then, the specimens were decalcified and histologically investigated with Hematoxylin eosin staining and Masson staining methods. Newly synthesized trabecular bone was inspected by image analysis system and the strength of bone defect area treated with graft-implantation was tested with biomechanical method-three point bending test.

RESULTSIn group I, trabecular bone was actively synthesized to generate a great amount of trabecular bone and osteon. Preliminary union and bone defect healing were completed with good biomechanical characteristics. There were no newly synthesized trabecular in the other three groups, and bone defect healing were not discovered. The amount of newly synthesized trabecular bone and the results of biomechanical testing differed significantly between group I and the other three (P < 0.01). The efficacy of group I was significantly better than that of the other three groups.

CONCLUSIONTrue bone ceramic complex composed with Cbfa1 gene modified rabbit skin fibroblasts can effectively heal bone defect in rabbits.

Animals ; Bone Regeneration ; Bone Substitutes ; Bone Transplantation ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; genetics ; Disease Models, Animal ; Fibroblasts ; cytology ; metabolism ; Plasmids ; genetics ; Rabbits ; Radius ; injuries ; physiopathology ; surgery ; Random Allocation ; Skin ; cytology ; Tissue Engineering ; methods ; Transfection

OBJECTIVETo evaluate the effects of two gastric cancer screening schemes for early detection of gastric cancer in a high-risk population.

METHODSA cluster random sampling method was used to select local residents aged 40-69 years from Linqu County, Shandong Province. "Serum pepsinogen initial screening combined with further endoscopic examination (PG scheme)" and "direct endoscopic examination (endoscopy scheme)" were conducted. The associations between screening schemes and detection rates of gastric cancer, and early gastric cancer/high-grade intraepithelial neoplasia were evaluated by unconditional logistic regression analysis.

RESULTSOverall, 3654 and 2290 participants completed PG and endoscopy schemes, respectively. A total of 11 (0.30%) cases of gastric cancer and 10 (0.27%) cases of high-grade intraepithelial neoplasia were detected by PG scheme, of which 7 (0.19%) cases were early gastric cancer. While, 19 (0.83%) cases of gastric cancer and 10 (0.44%) cases of high-grade intraepithelial neoplasia were detected by endoscopy scheme, with 12 (0.52%) cases of early gastric cancer. Compared with the PG scheme, the endoscopy scheme had a significantly higher detection rates of gastric cancer (OR = 2.83, 95%CI 1.34-5.98), and early gastric cancer/high-grade intraepithelial neoplasia (OR = 2.12, 95%CI 1.12-4.02).

CONCLUSIONSThe endoscopy scheme is more effective in the detection of gastric cancer in a high-risk population, particularly for early gastric cancer/high-grade intraepithelial neoplasia than the PG scheme.

Adult ; Aged ; Carcinoma ; blood ; diagnosis ; Carcinoma in Situ ; blood ; diagnosis ; Early Detection of Cancer ; methods ; Female ; Gastroscopy ; Humans ; Male ; Mass Screening ; methods ; Middle Aged ; Pepsinogen A ; blood ; Stomach Neoplasms ; blood ; diagnosis